Functional reconstitution and characterization of AqpZ, the E. coli water channel protein

Borgnia, Mario J.; Kozono, David; Calamita, Giuseppe; Maloney, Peter C.; Agre, Peter

doi:10.1006/jmbi.1999.3032

Cited by 287 publications

(382 citation statements)

References 33 publications

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“…Nevertheless, ultrastructural studies confirmed that AQP1 is tetrameric in the plasma membranes of transfected cells (22) and in reconstituted membrane crystals (23,24). AqpZ forms particularly stable tetramers which remain noncovalently associated even in SDS (1). It was therefore surprising that GlpF was reported to be a monomer when analyzed by velocity sedimentation in nondenaturing detergent (14) and by freeze-fracture electron microscopy of oocyte membranes (15).…”

Section: Discussionmentioning

confidence: 99%

“…Members of this protein superfamily are found throughout nature, and virtually all microorganisms have at least one member. E. coli has two, AqpZ and GlpF, but despite their high degree of sequence similarity, recent publications have noted major differences in oligomeric structure (1,15), which may be linked to substrate specificity (12,15).…”

Section: Discussionmentioning

confidence: 99%

“…Escherichia coli contains two members of this family: AqpZ, which is selectively permeated by water (1), and GlpF, the facilitator of glycerol transport (2). AqpZ and GlpF are representative of an early divergence, since water is essential for cellular hydration, and glycerol is necessary for cellular nutrition and osmotic balance.…”

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confidence: 99%

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Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

Borgnia

Agre

2001

Proc. Natl. Acad. Sci. U.S.A.

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195

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A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg 2؉ stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl2. Our studies demonstrate that, unlike AqpZ, a waterselective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

Borgnia

Agre

2001

Proc. Natl. Acad. Sci. U.S.A.

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192

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“…Other biological membranes, such as those in mammalian optic lenses (2), erythrocytes (3), and many other cell membranes (4) are constitutively AQP-rich. The permeabilities of AQP-rich membranes are orders of magnitude higher than those observed for unmodified phospholipid membranes (5). Additionally, some members of the AQP family have excellent solute retention capabilities for small solutes such as urea, glycerol, and glucose, even at high water transport rates (5,6).…”

mentioning

confidence: 97%

“…The permeabilities of AQP-rich membranes are orders of magnitude higher than those observed for unmodified phospholipid membranes (5). Additionally, some members of the AQP family have excellent solute retention capabilities for small solutes such as urea, glycerol, and glucose, even at high water transport rates (5,6). These properties result from the unique structure of the water-selective AQPs.…”

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confidence: 98%

Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z

Kumar

Grzelakowski

Zilles

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

666

643

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The permeability and solute transport characteristics of amphiphilic triblock-polymer vesicles containing the bacterial water-channel protein Aquaporin Z (AqpZ) were investigated. The vesicles were made of a block copolymer with symmetric poly-(2-methyloxazoline)-poly-(dimethylsiloxane)-poly-(2-methyloxazoline) (PMOXA 15-PDMS110-PMOXA 15) repeat units. Light-scattering measurements on pure polymer vesicles subject to an outwardly directed salt gradient in a stopped-flow apparatus indicated that the polymer vesicles were highly impermeable. However, a large enhancement in water productivity (permeability per unit driving force) of up to Ϸ800 times that of pure polymer was observed when AqpZ was incorporated. The activation energy (E a) of water transport for the protein-polymer vesicles (3.4 kcal/mol) corresponded to that reported for water-channel-mediated water transport in lipid membranes. The solute reflection coefficients of glucose, glycerol, salt, and urea were also calculated, and indicated that these solutes are completely rejected. The productivity of AqpZ-incorporated polymer membranes was at least an order of magnitude larger than values for existing salt-rejecting polymeric membranes. The approach followed here may lead to more productive and sustainable water treatment membranes, whereas the variable levels of permeability obtained with different concentrations of AqpZ may provide a key property for drug delivery applications.permeability ͉ triblock copolymer ͉ water treatment B iological membranes have excellent water transport characteristics, with certain membranes able to regulate permeability over a wide range. The permeability of membranes such as those present in the proximal tubules of the human kidney (1) can be increased by insertion of specific water-channel membrane proteins known as Aquaporins (AQPs). Other biological membranes, such as those in mammalian optic lenses (2), erythrocytes (3), and many other cell membranes (4) are constitutively AQP-rich. The permeabilities of AQP-rich membranes are orders of magnitude higher than those observed for unmodified phospholipid membranes (5). Additionally, some members of the AQP family have excellent solute retention capabilities for small solutes such as urea, glycerol, and glucose, even at high water transport rates (5, 6). These properties result from the unique structure of the water-selective AQPs. These AQPs have six membrane-spanning domains and a unique hourglass structure (7) with conserved charged residues that form a pore that allows both selective water transport and solute rejection. The AQP used in this study was a bacterial aquaporin from Escherichia coli, Aquaporin Z (AqpZ). AqpZ was selected because it can enhance the permeability of lipid vesicles by an order of magnitude while retaining small uncharged solutes (5). Additionally, AqpZ can be expressed in relatively large quantities in E. coli and has been reported to be quite stable under different reducing conditions and at temperatures of 4°C (5)-properties that make it att...

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Single Proteins Observed by Atomic Force Microscopy

et al. 2001

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Here we discuss the experimental approaches that have allowed high resolution atomic force microscopy (AFM) imaging, and review results that show AFM to be of great interest for biologists, AFM allows single proteins to be imaged under physiologically relevant conditions. The exceptional signal-to-noise ratio and resolution of AFM topographs enables the oligomerization state and characteristic substructures of individual proteins to be resolved. Several examples demonstrate the capabilities of AFM to directly observe single proteins, and their conformational changes, to study protein-protein interactions and to follow the assembly of membrane proteins. We consider the AFM techniques that have allowed high resolution imaging, and review results that show AFM to be a powerful method to analyze biological processes at the level of single molecules.

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Functional reconstitution and characterization of AqpZ, the E. coli water channel protein

Cited by 287 publications

References 33 publications

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z

Single Proteins Observed by Atomic Force Microscopy

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